Testing of blood samples to detect target nucleic acids can be challenging. The sensitivity and specificity of nucleic acid-based blood assays are influenced not only by the abundance of the nucleic acid, but by its availability for amplification and detection. For example, the availability may be affected if the nucleic acid is sequestered in viral capsids or if it is associated with bound proteins. More importantly, the components of blood cells have well-known interfering effects on nucleic acid diagnostic techniques, such as inhibition of amplification and signal detection. A number of solutions have been developed to deal with this problem.
Most nucleic acid detection systems require a pre-extraction of total nucleic acids in samples before application to the amplification and detection system. Some nucleic acid extraction protocols involve lysing all blood cells, binding nucleic acids, and successfully washing away inhibitory substances. Alternatively, whole blood can be centrifuged to remove red and white blood cells from plasma, separating the cells from plasma-borne nucleic acids and pathogens. Plasma separation performed in this manner works well but is not ideal for large numbers of samples and increases contamination risks due to additional blood handling steps. Simpler methods for pre-processing lysed blood samples for direct nucleic acid analysis have been developed. Generally, small amounts of blood are lysed in reagents that sequester inhibitory substances. These specially-treated blood samples are then used in small amounts in amplification reactions containing specialized buffers and inhibition-tolerant enzymes. However, the enzymes required for these techniques are more expensive than standard amplification enzymes. Furthermore, there are stricter limitations on the amount of sample and the methods that can be used to analyze these preparations, possibly reducing assay sensitivity.
Given these disadvantages, and the increasing demand for inexpensive consumable detection methods particularly in remote diagnostic medicine or at sites with minimally-skilled technical personnel, the need exists for simple Point-of-Care (POC) tools for the detection of nucleic acids in blood. Such ideal detection devices for blood should be able to receive whole blood or minimally diluted whole blood; eliminate or segregate inhibitory substances away from the amplification and detection reagents; and operate quickly, inexpensively, and in a fully integrated system with minimal requirements for user or instrument intervention.